The last fifty years has brought profound advances in the field of clinical microbiology and the treatment and management of infectious diseases. However, life threatening and debilitating systemic and localized microbial infections remains a major healthcare problem. Mortality resulting from infectious agents remains particularly high among infants, the elderly, the immunosuppressed, patients in long-term care facilities, and skilled nursing homes, Moreover, the emergence of multi-drug resistance organisms such as vancomycin resistant enterococci (VRE) and methacillin resistant Staphylococcus aureaus (MRSA) have increased the challenges of caring for hospitalized patients. Hospital acquired infections (nosocomial infections) caused by organisms such as VRE, MRSA and pseudomonas add significantly to patient suffering, increased hospital stays, iatrogenic mortality, and increased healthcare costs.
Inadequately or improperly treated microbial infections are largely responsible for the rise of multiple drug resistant strains of bacteria that cause many nosocomial infections. Drug resistance, specifically antibiotic resistance, often occurs when the antibiotic used to treat an infection is either improperly selected, prescribed in a fashion that does not effectively eradicate the infectious agent, or as a result of poor patient compliance. Furthermore, when ineffective or unnecessary antibiotics are prescribed any infecting bacteria present continues to multiply unabated often resulting in life threatening complications necessitating expensive, aggressive treatments including otherwise needless hospitalization. Therefore, the accurate and rapid diagnosis of a potential infectious agent is critical to improved patient care, reduced healthcare costs and the preservation of antimicrobial efficacy.
The first step in the proper diagnosis of a microbial infection is the determination of the causative agent. Although it is common and acceptable protocol to treat high risk patients with a broad spectrum antibiotic based on clinical judgment prior to establishing the existence, identity and susceptibility pattern of a putative infectious agent, it is still essential that such testing be conducted and treatment changed or modified as indicated by the test results.
Generally speaking, when an infection is suspected, samples are taken from the affected site and analyzed using staining techniques, genetic based assays such as the polymerase chain reaction (PCR) test and cultures. Stains can provide a skilled microscopist with reliable information about the morphology of any microorganism present and the type of cell infected. However, stains are largely non-specific and seldom definitive. Polymerase chain reaction assays are highly specific and definitive when samples are free of PCR inhibitors, contamination and contain near pure cultures of the infectious agent. However, most clinical samples taken directly from infected patients are contaminated and contain PCR inhibitors. Furthermore, PCR assays are expensive, highly specialized, and require multiple probes in order to identify an unknown organism. Consequently, PCR is seldom, if ever used to replace standard microbial culture and identification techniques as the front line test in the clinical microbiology laboratory.
While it is possible for viruses, fungi, bacteria and occasionally other microscopic life forms to cause clinical infections, the remainder of this discussion will focus on bacterial infections. This is not intended to limit the techniques and procedures disclosed herein to bacteria, but rather in deference to brevity.
Standard culture techniques rely on the use of solid nutrient media on which a clinical sample is placed, then physically diluted by spreading the sample over the solid media's surface using a sterile inoculation device such as a wire shaft terminating in a loop. The dilution process, colloquially known as “streaking,” facilitates the isolation and subsequent identification of the infectious agent by permitting single bacterial cells to be separated from others present in the sample. The diluted specimen gives rise to individual cells that multiply to form a population of homogeneous prodigy organisms. The resulting population of homogeneous organisms is referred to as a colony. Samples containing only one colony type are known as pure cultures, hence, a pure culture is a population of the same species of microorganism.
It is essential that only pure cultures be tested for identity and drug susceptibility so that there are no synergistic or antagonistic effects that could result in erroneous conclusions. After a microorganism has been isolated, usually 24 to 48 hours post-inoculation, the microbiologist must identify the organism and test it for antimicrobial susceptibility.
The first step in identifying bacteria is to broadly classify the organism into one of two classes: Gram positive or Gram negative. This process is performed using a simple staining procedure or through the astute judgment of an experienced clinical microbiologist. Based on this initial classification, the microbiologist selects a panel of biochemical tests to identify the organism and chooses the panel of antimicrobials best suited to test the organism's drug susceptibility.
Traditionally, bacteria are identified by inoculation of a series of tubes containing growth media, a specific substrate, such as a sugar, and an indicator system that responds demonstrably to any affirmative action of the microorganism on the substrate. For example, if a bacteria can enzymatically degrade sugar present in a culture media, acidic by-products from the metabolism of the sugar will drop the pH of the media. This drop in pH (the acidification of the media), is detected by a change in color of a pH indicator added to the media. For example, phenol red changes from red to yellow in the presence of acid pH. These traditional growth dependent techniques require a minimum of 18-24 hours before results can be determined. Moreover, the labor and material costs associated with such methods are high, consequently only a limited number of biochemical substrates are used in the initial screen. If the results obtained after the first 18-24 hours of incubation are inconclusive, additional tests must be conducted which further delay definitive results in reaching the physician.
At the time the initial identification scheme is inoculated, the microbiologist usually initiates anti-microbial susceptibility testing. Traditionally, antimicrobial susceptibility testing is performed using liquid growth media containing pre-determined concentrations of selected antibiotics, or the surface of a solid growth media is inoculated such that a confluent lawn of bacteria will develop and small disks containing an antibiotic are placed on the surface of the solid media. The liquid culture media technique is known as the broth dilution method and the inoculated solid agar procedure is referred to as the Kirby-Bauer test.
In the broth dilution test an organism is considered sensitive to the concentration of drug in the tube if the organism fails to grow in the tube, or tubes, containing the drug and thrives in a media tube with no drug present (growth control tube). There are basically two types of broth dilution susceptibility assays routinely used. One is referred to as a minimum inhibitory concentration assay (MIC), and the other is referred to as a minimum bactericidal concentration (MBC) assay. In the MIC assay an inhibitory anti-microbial compound such as an antibiotic is serially diluted (usually two-fold) in culture medium. The concentration of the drug in the last dilution where the organism fails to grow (the last no growth culture) is referred to as the minimum inhibitory concentration for that compound. A MBC assay is performed in exactly the same fashion except that the anti-microbial compounds used are known to exert a killing, rather than an inhibitory effect on an organism. An organism is considered sensitive to an anti-microbial compound when its MIC or MBC is less than an established minimum. For example, Staphylococcus aureus is considered sensitive to penicillin when it fails to grow in a culture containing 0.12 micrograms per milliliter or less of penicillin in the test media and resistant if the organism thrives in media containing 0.25 micrograms of penicillin per milliliter or more.
When the Kirby-Bauer is used an organism is considered sensitive to an anti-microbial compound If a zone of no growth equal to or exceeding an established minimum diameter is present around the drug-containing disk. Both the Kirby-Bauer and broth dilution methods are laborious to set up, require skilled personnel to interpret and require a minimum of 18-24 hours before results are available. Therefore, techniques and equipment that could decrease cost, increase accuracy, provide faster, more reliable results to the Physician would significantly improve patient care and reduce inappropriate and unnecessary antimicrobial therapy.
In the 1980's a variety of new technologies were introduced to clinical microbiology designed to address a number of the aforementioned problems. Most of these systems relied on conventional biochemical tests and broth dilution drug susceptibility assays that had been miniaturized and combined into single multi-well trays or plates (microtiter plates). In the miniaturized configuration each 15 mL tube of biochemical or antimicrobial used in the traditional assay was reduced in scale to a volume of approximately 200 μL. Consequently, microtiter plates containing up to 96 individual “wells” would represent 96 individual tubes in the traditional format. This resulted in an overall reduction in processing time and materials cost, but still relied on visible growth of the putative pathogen that required a minimum of 18-24 hours post-inoculation. Moreover, skilled personnel were required to read the plates. The miniaturization of the biochemical assays and drug-containing tubes from 15 mL to 200 μL resulted in difficulty reading plates because visible growth is often barely detectable. Consequently, much of the time and cost savings associated with the inoculation and set-up was lost reading and reporting results.
In an effort to increase reliability and decrease reading and reporting times associated with antimicrobial susceptibility testing, automated reading devices were developed which could detect the presence or absence of growth in a well by reading the turbidity using a spectrophotometer and comparing it to control wells. (See U.S. Pat. Nos. 4,448,534 and 3,957,583).
In addition to turbidimetric methods for determining the growth of a microorganism other techniques such as colorimetric detection systems (see U.S. Pat. No. 5,817,475) light detection and light scattering systems (see U.S. Pat. Nos. 4,118,280; 4,116,775; 3,942,899; 3,832,532; 3,901,588 and 3,928,140), pH measurements systems (see U.S. Pat. No. 5,496,697), and fluorometric and nephelometric detection systems (see U.S. Pat. No. 4,784,947) were developed. Another automated instrument for reading antimicrobial microtiter plates uses a fluorescent procedure which reads the fluorescence emitted as the result of bacterial enzymatic action on a fluorogenic substrate (see EPO 091,837 B).
In addition to systems designed to merely automate the reading and reporting steps, several different automated formats have been developed to completely automate the inoculation, incubation, reading and reporting steps associated with antimicrobial testing. Examples of such automated devices can be found in U.S. Pat. Nos. 5,645,800; 5,518,686; 4,676,951 and 4,681,741. Present automated methods for the inoculation, incubation, reading and reporting of antimicrobial susceptibility and bacterial identification have significantly advanced the reproducibility and accuracy of these assays and has decreased cost and increased throughput for the clinical laboratory. However, the aforementioned technologies all rely on a single endpoint determination based on pre-set parameters. This is especially problematic when fluorogenic substrates are used. Growth rates between different species of organisms as well as between different strains of the same species are common. In certain cases the growth rate may lag sufficiently so that when a pre-determined reading interval is reached there will be insufficient growth, and hence fluorescence to be detectable. As a result, the well will be read by the instrument as “no growth” indicating susceptibility to the respective drug at the level being tested. This in turn could be reported to the clinician that would initiate antimicrobial therapy based on an erroneous result.
Therefore, there is a need for a clinical microbiology test platform that combines anti-microbial susceptibility testing with microorganism identification. Moreover, there is a need for a combined system that generates accurate, reproducible results that are available quickly and with lower materials and labor cost than presently available.